von Willebrand Factor (vWF) - Cleaving Protease

ABSTRACT

This invention is intended to isolate and identify a vWF-specific cleaving protease. The vWF-specific cleaving protease cleaves a bond between residues Tyr 842 and Met 843 of vWF and comprises a polypeptide chain having Leu-Leu-Val-Ala-Val (SEQ ID NO: 1) as a partial sequence, and more preferably comprises a polypeptide chain having the partial N-terminal amino acid sequence of a mature protein, Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val (SEQ ID NO: 2), and having a molecular weight of 105 to 160 kDa in SDS-PAGE under reducing or non-reducing conditions. Isolation and identification of this vWF-specific cleaving protease have led to the possibility of replacement therapy for patients having diseases resulting from a deficiency of the protease, such as thrombotic thrombocytopenic purpura.

This application is a Divisional Application of U.S. Ser. No.12/103,899, filed Apr. 16, 2011, which is a Divisional Application ofU.S. Ser. No. 11/296,294, filed Dec. 8, 2005 Issued U.S. Pat. No.7,361,748, issue date Apr. 22, 2008, which is a Divisional Applicationof U.S. Ser. No. 10/475,538, filed Oct. 22, 2003, Issued U.S. Pat. No.7,112,666 issue date Sep. 26, 2006, which is a 371 application ofPCT/JP02/04141, filed Apr. 25, 2002, which claims priority from Japanesepatent applications JP 2001-128342, filed Apr. 25, 2001, JP 2001-227510,filed Jul. 27, 2001, JP 2001-302977, filed Sep. 28, 2001 and JP2002-017596, filed Jan. 25, 2002. The entire contents of each of theaforementioned applications are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a plasma protein related to the fieldof medical drugs. More particularly, the present invention relates to aprotease that specifically cleaves von Willebrand factor (it may behereafter referred to as “vWF”), which is associated with bloodcoagulation. The vWF-cleaving protease of the present invention enablesreplacement therapy for patients with diseases resulting from defects ordecreases in this protease, such as thrombotic thrombocytopenic purpura(it may be hereafter referred to as “TTP”). In addition, the use thereofas a novel antiplatelet thrombotic agent is expected.

BACKGROUND ART

vWF is produced in vascular endothelial cells or megakaryocytes, and isa blood coagulation factor in which a single subunit comprising 2,050amino acid residues (monomers of about 250 kDa) are bound by an S—S bondto form a multimer structure (with a molecular weight of 500 to 20,000kDa). The level thereof in the blood is about 10 μg/ml, and ahigh-molecular-weight factor generally has higher specific activity.

vWF has two major functions as a hemostatic factor. One of the functionsis as a carrier protein wherein vWF binds to the blood coagulationfactor VIII to stabilize it. Another function is to form platelet plugby adhering and agglomerating platelets on the vascular endothelialsubcellular tissue of a damaged vascular wall.

Thrombotic thrombocytopenic purpura is a disease that causes plateletplug formation in somatic arterioles and blood capillaries throughoutthe whole body. In spite of recent advances in medical technology, themorbidity associated with this disease approximately tripled from 1971to 1991. Pathologically, TTP is considered to result from vascularendothelial cytotoxicity or vascular platelet aggregation.Immunohistologically, a large amount of vWFs are recognized in theresulting platelet plugs, and vWF is considered to play a major role incausing them. A normal or high-molecular-weight vWF multimer structureis dominant in a TTP patient, and an unusually large vWF multimer(ULvWFM) or large vWF multimer (LvWFM) is deduced to play a major rolein accelerating platelet aggregation or microthrombus formation underhigh shearing stress. In contrast, vWF was known to degrade at aposition between residues Tyr 842 and Met 843 by the action ofvWF-cleaving protease in the circulating blood of a healthy person underhigh shearing stress. Accordingly, TTP is considered to occur in thefollowing manner. The protease activity in the plasma is lowered forsome reason, and ULvWFM to LvWFM are increased to accelerate plateletaggregation. This forms platelet plugs in blood vessels.

Recently, Furlan et al. (Blood, vol. 87, 4223-4234: 1996, JP PatentPublication (Kohyo) No. 2000-508918) and Tsai et al. (Blood, vol. 87,4235-4244: 1996) developed a method for assaying vWF-specific cleavingprotease. In their report, this protease activity was actually loweredin TTP. The aforementioned authors reported that this enzyme wasmetalloprotease in the plasma and partially purified. However, they havenot yet succeeded in the amino acid sequencing which would specify theprotease. There have been no further developments since then.

DISCLOSURE OF THE INVENTION

Up to the present, plasmapheresis therapy has been performed fortreating patients who congenitally lack vWF-specific cleaving proteaseand patients who had acquired positive antibodies against this protease.Establishment of replacement therapy using purified products or a puresubstance such as a recombinant gene product of the aforementionedprotease is desired. Familial TTP patients congenitally lackvWF-specific cleaving protease, and non-familial TTP is caused byposteriori production of autoantibodies against the aforementionedprotease. Accordingly, replacement therapy for this protease ispreferable for familial TTP patients (plasma administration is actuallyperformed), and removal of autoantibodies by plasmapheresis andsubstitution of this protease are necessary for non-familial TTP.Further, the use of this protease as a novel antiplatelet thromboticagent can also be expected.

As mentioned above, however, Furlan et al. (Blood, vol. 87, 4223-4234:1996, JP Patent Publication (Kohyo) No. 2000-508918) and Tsai et al.(Blood, vol. 87, 42354244: 1996) have suggested that the vWF-cleavingprotease was metalloprotease in the plasma. It was reported to bepartially purified, and concentrated 1,000- to 10,000-fold from theplasma in terms of its specific activity. Even under these conditions,there has been no advancement in the analysis of the properties of thisprotease, such as the amino acid sequence of its protein, over theperiod of roughly 5 years that has passed since then. No specificbiological information has yet been obtained regarding this protease. Asreported by Furlan et al., the protein of interest is supposed to begigantic, and there may be various problems associated therewith. Forexample, diversified forms of this protease, such as various interactingmolecules or cofactors, are expected. Based on the complexity ofpurification processes, deteriorated capacity of separation bynonspecific interaction during the purification step, and other factors,it is deduced to be very difficult to isolate and identify the proteasefrom a plasma fraction by the purification process according to Furlanet al.

Under the above circumstances, the present inventors have conductedconcentrated studies in order to isolate and identify the vWF-cleavingprotease. As a result, they have succeeded in isolating and purifyingthe vWF-cleaving protease of interest, which had not yet been reported.Thus, they have succeeded in identifying an amino acid sequence of themature protein and a gene encoding this amino acid sequence.

The vWF-cleaving protease of the present invention can cleave a bondbetween residues Tyr 842 and Met 843 of vWF. According to oneembodiment, this protease has a molecular weight of 105 to 160 kDa or160 to 250 kDa in SDS-PAGE under reducing or non-reducing conditions. Itis comprised of a polypeptide chain having Leu-Leu-Val-Ala-Val as apartial sequence. More preferably, it is comprised of a polypeptidechain having the partial N-terminal amino acid sequence of a matureprotein, i.e., Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val.It is a novel substance characterized by the following properties.

1) vWF-Cleaving Activity

According to the N-terminal sequence analysis of the cleavage fragment,the protease of the present invention cleaves a peptide bond betweenresidues Tyr 842 and Met 843.

2) Fractionation by Gel Filtration

When fractionation is performed by gel filtration chromatography usingFI paste as a starting material, most activities are collected in afraction with a molecular weight of 150 to 300 kDa. According to oneembodiment of the present invention, an actually obtained activesubstance is found to have a molecular weight of about 105 to 160 kDa inelectrophoresis. Accordingly, the protease of the present invention is asubstance that is likely to form a dimer or the like or to bind toanother molecule or a substance that can be easily degraded or can havea heterogeneous sugar chain added.

3) Ammonium sulfate precipitation

For example, when FI paste is used as a starting material, a largeportion of this protease is recovered as a precipitation fraction from aroughly purified fraction with the use of 33% saturated ammoniumsulfate.

4) SDS-PAGE

For example, the protease of the present invention derived from FI pasteprepared from pooled human plasma or cryoprecipitate mainly has amolecular size of about 105 to 160 kDa determined by a molecular weightmarker in SDS-PAGE. Based on the nucleic acid sequence as shown in SEQID NO: 15, when an amino acid sequence represented by a frame between anatg initiation codon at position 445 and a tga termination codon atposition 4726 is expressed by gene recombination, there are somevariations in molecular sizes depending on a host. However, a molecularsize of about 160 to 250 kDa determined by a molecular weight marker isexhibited. This size is observed in the plasma of healthy humans and inthat of some TTP patients. Several molecular species of this proteaseare present in human plasma, caused by the presence of alternativesplicing products (SEQ ID NOs: 16 to 21) recognized at the time of genecloning, differences in post-translational modification such as sugarchain addition, or degradation during purification. Further, thisprotease could be partially recovered in an active state after SDS-PAGEunder non-reducing conditions.

5) Analysis of Amino Acid Sequence

The amino acid sequence of the isolated polypeptide fragment wasanalyzed. This presented an example of a polypeptide chain having asequence Leu-Leu-Val-Ala-Val (SEQ ID NO:1) as a partial amino acidsequence and a sequenceAla-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val (SEQ ID NO: 2)as a N-terminal amino acid sequence of a mature protein. Further, withcurrent bioinformatics (BIOINFORMATICS: A Practical Guide to theAnalysis of Genes and Proteins, edited by Andreas D. Baxevanis and B. F.Francis Ouellette), a nucleic acid sequence encoding the amino acidsequence was highly accurately identified by searching a database basedon the aforementioned partial sequence. More specifically, the genomedatabase was searched by the tblastn program. This identified achromosome clone (AL158826) that is deduced to encode the protease ofthe present invention. Further, clones (AI346761 and AJ011374) that arededuced to be a part of the protease of interest and a part of thepolypeptide to be encoded by the aforementioned genome were identifiedthrough collation with the Expressed Sequence Tag (EST) database. Basedthereon, the amino acid sequence as shown in SEQ ID NO: 3 or 7 wasidentified as an active vWF-cleaving protease site.

GCT GCA GGC GGC ATC CTA CAC CTG GAG CTG CTG GTG GCC GTG (SEQ ID NO: 5),a sequence deduced from the genome, and more preferably CTG CTG GTG GCCGTG (SEQ ID NO: 4), a portion thereof, the transcriptome of which wasconfirmed by EST, was obtained. The obtained nucleotide sequence wasanalyzed, and motif analysis was carried out based on the deducedsequence. As a result, it was found to have a metalloprotease domain asa candidate for the protease of the present invention. Based on theabove findings, it became possible to disclose a sequence of apolypeptide chain as a more specific example of the protease. Also,activities of proteases are generally known to vary depending on, forexample, substitution, deletion, insertion, or introduction of pointmutation into a portion of the amino acid sequence (Blood coagulationfactor VII mutants, Soejima et al., JP Patent Publication (Kokai) No.2001-61479 A). Similarly, the protease of the present invention can bemodified by, for example, deletion, substitution, or addition of one orseveral amino acids, to prepare optimized proteases.

The protease proteins were further mass-produced, and 29 amino acidsequences from the N-terminus were determined. These amino acidsequences are shown in SEQ ID NO: 8. This result is substantially thesame as the sequence as shown in SEQ ID NO: 3 or 7 deduced bybioinformatics. Only one difference is that the amino acid 27th in SEQID NO: 3 or 7 was Glu while it was Arg according to the present analysisof the N-terminal sequence. This was considered to be a genepolymorphism. Thus, this protease was confirmed to be comprised of apolypeptide chain having the amino acid sequence as shown in SEQ ID NO:3 or 7 at its N-terminus as a mature unit. A gene fragment encoding thisprotease was then cloned in the following manner.

Based on the nucleic acid sequence as shown in SEQ ID NO: 7, a senseprimer (SEQ ID NO: 9) and an antisense primer (SEQ ID NO: 10) wereprepared based on the nucleic acid sequence underlined in FIG. 9, and agene sandwiched between these primers was amplified. This fragment wascloned, and the nucleotide sequence was then confirmed. This fragmentwas used as a probe for Northern blotting to analyze the site at whichthe protease gene was expressed. As a result, this protease gene wasfound to be expressed mainly in the liver. Accordingly, the human livercDNA library was purchased, and a gene encoding this protease wasidentified using a rapid amplification of cDNA ends (RACE) technique.Based on these results, in the case of the largest sequence ofapproximately 5 kb of mRNA (cDNA) reaching the poly(A) addition site asshown in SEQ ID NO: 15 was identified.

Based on the amino acid sequence deduced from this gene sequence, thisprotease was deduced to have a preprosequence, and to belong to thedisintegrin and metalloprotease (ADAM) family having a disintegrin-likedomain, a metalloprotease domain, and the like, and particularly to theADAM-TS family having a thrombospondin Type-1 (TSP-1) domain. Finally,including those having insertion or deletion in a part of the nucleicacid sequence, isoforms as shown in SEQ ID NOs: 16 to 21 havingsequences as shown in SEQ ID NOs: 3 and 7 at the N-terminuses after themature preprosequence has been cleaved were identified. Thus, theprotease of the present invention should cleave vWF between residues Tyr842 and Met 843 and should have the Leu-Leu-Val-Ala-Val (SEQ ID NO: 1)sequence as a partial amino acid sequence.

The vWF-cleaving protease of the present invention can be generallyprepared by the following process.

According to the present invention, a process for assaying the proteaseactivity is characterized by the possibility of evaluating activitywithin a short period of time. According to the report by Furlan et al.(Blood, vol. 87, 4223-4234: 1996, JP Patent Publication (Kohyo) No.2000-508918 A), activity is assayed by analyzing vWF-cleaving patternsby Western blotting using the anti-vWF antibody, and thus, it takes timeto transfer the protease to a filter. More specifically, this processrequires approximately at least 45 hours in total, i.e., 24 hours forthe enzymatic reaction with a substrate vWF, 17 hours forelectrophoresis, and 3 hours to transfer the protease to a filter,followed by detection using the anti-vWF antibody. In contrast, thepresent inventors completed activity assay in 18 hours in total, i.e.,16 hours for the enzymatic reaction with a substrate vWF, and 2 hoursfor electrophoresis and detection. This indicates that the time requiredfor the assay can be reduced to one third or less of that required forthe conventional assay. This can also shorten the time required for thepurification process, and in turn can lower the degree of the proteaseto be inactivated. Accordingly, purification efficiency is improvedcompared with that attained by the method of Furlan et al., and as aresult, the degree of purification is also enhanced.

Further, the starting material was examined using the aforementionedassay system. As a result, it was found that the protease activity wasmore concentrated in FI paste than in the cryoprecipitate that had beenreported by Furlan et al. in the past. FI paste was used as a startingmaterial, and the aforementioned rapid activity assay systems werecombined. This enabled isolation and identification of the protease ofinterest. In a specific embodiment, a purification process combining gelfiltration chromatography with ion exchange chromatography is employed,and the aforementioned activity assay system is also combined.

More specifically, FI paste is solubilized with a buffer, and theresultant is fractionated by gel filtration chromatography. The proteaseactivity is fractionated at the elution region with a molecular weightof 150 to 300 kDa deduced from the size marker of gel filtration.Thereafter, the resultant is precipitated and concentrated using 33%saturated ammonium sulfate. This procedure is repeated three times intotal. The active fraction obtained in the third gel filtration ispooled, and the resultant is subjected to dialysis at 4° C. overnightwith a buffer comprising 50 mM NaCl added to 50 mM Tris-HCl (pH 7.1).Thereafter, the dialysis product is subjected to anion exchangechromatography (DEAE) and eluted stepwise with 0.25 M NaCl. The presentinventors have conducted concentrated studies in order to find a processfor isolating and identifying the protease of the present invention. Asa result, they found that, surprisingly, the protease was recoverable asan active band after non-reducing SDS-PAGE. In order to achieve furthermass production, the purified and concentrated fraction was applied tothe Biophoresis utilizing the principle of SDS-PAGE. Thus, a fractionhaving vWF-cleaving activity was isolated from the electrophoresedfraction. According to the approximate calculation of the specificactivity up to this phase, purification of about 30,000- to 100,000-foldwas achieved. This procedure was efficiently and rapidly repeatedseveral times, and thus, about 0.5 μmole of sample that is the currentlimit of the analysis of amino acid sequence was obtained. Thus,analysis of amino acid sequence became feasible. More specifically, afinal step of separation and purification (Biophoresis) based on theprinciple of SDS-PAGE is important, and it is based on the findings as aresult of concentrated studies, which had led to the completion of thepresent invention.

According to the report by Furlan et al., specific activity was improvedby as much as about 10,000 times, although the protease was notsubstantially isolated or identified. This could be because ofdeactivation during purification or the difficulty of isolating andidentifying molecules, which were gigantic proteins capable ofinteracting with various other proteins such as the protease of thepresent invention by a separation method utilizing various types ofliquid chromatography. Further, the protease content in the plasma wasdeduced to be very small, and thus, it was necessary to await theestablishment of the process according to the present invention.Furthermore, the use of this process enables the purification ofrecombinant genes.

Based on the findings of the present invention, peptides or proteinsprepared from the obtained sequences are determined to be antigens. Withthe use thereof, a monoclonal antibody, a polyclonal antibody, or ahumanized antibody thereof can be prepared by general immunizationtechniques (Current Protocols in Molecular Biology, AntibodyEngineering: A PRACTICAL APPROACH, edited by J. McCAFFERTY et al. orANTIBODY ENGINEERING second edition, edited by Carl A. K. BORREBAECK).Alternatively, an antibody that binds to the aforementioned protein canbe prepared by antibody-producing techniques utilizing phage display(Phage Display of Peptides and Proteins: A Laboratory Manual, edited byBrian K. Kay et al., Antibody Engineering: A PRACTICAL APPROACH, editedby J. McCAFFERTY et al. or ANTIBODY ENGINEERING second edition, editedby Carl A. K. BORREBAECK). Alternatively, based on these techniques, aneutralizing antibody acting against the protease activity or a simplebinding antibody can be isolated from a specimen from a TTP patient whohas an autoantibody positive against this protease. These antibodies canbe applied to diagnosis and therapy of diseases such as TTP.

Based on the obtained genome or EST sequence, cDNA or a genomic geneencoding the protease of the present invention can be cloned by a commontechnique (Molecular Cloning, 2nd edition). Further, bioinformaticstechniques (BIOINFORMATICS: A Practical Guide to the Analysis of Genesand Proteins, edited by Andreas D. Baxevanis and B. F. FrancisOuellette) enable cloning of the proteins of other animal species thatare homologous thereto, and the resultant gene is fractured by a commontechnique (for example, Gene Targeting: A Practical Approach, FirstEdition, edited by A. L. Joyner, Teratocarcinomas and embryonic stemcell a practical approach) to produce TTP-like animal models. Inparticular, the identification of the gene sequence encoding the proteinderived from a mouse enables the production of a knockout mouse havingthis gene. Thus, a disease mouse model of congenital TTP or the like canbe prepared.

In accordance with a common technique (for example, J. Sambrook et al.,Molecular Cloning, 2nd edition, or CURRENT PROTOCOLS IN MOLECULARBIOLOGY), these genes are incorporated into a suitable expressionvector, the resultant is transformed into a suitable host cell, and thegene recombinant product of the protease can be thus prepared. In thiscase, the gene to be incorporated is not necessarily the one thatencoded the entire region of the protein. It also includes a partialexpression of the protein as defined by a domain depending on its usage.

For example, the polynucleotide according to the present invention isintroduced into a host cell using a conventional technique such astransduction, transfection, or transformation. The polynucleotide isintroduced solely or together with another polynucleotide. Anotherpolynucleotide is introduced independently, simultaneously, or incombination with the polynucleotide of the present invention.

For example, the polynucleotide of the present invention is transfectedin a host cell, such as a mammalian animal cell, by a standard techniquefor simultaneous transfection and selection using another polynucleotideencoding a selection marker. In this case, the polynucleotide would begenerally stably incorporated in the genome of the host cell.

Alternatively, the polynucleotide may be bound to a vector comprising aselection marker for multiplication in a host. A vector construct isintroduced to a host cell by the aforementioned technique. In general, aplasmid vector is introduced as DNA of a precipitate, such as a calciumphosphate precipitate, or a complex with a charged lipid.Electroporation is also employed for introducing the polynucleotide intoa host. When the vector is a virus, this virus is packaged in vitro orintroduced into a packaging cell, thereby introducing the packaged virusinto a cell.

Extensive techniques that are suitable for producing a polynucleotideand introducing the resulting polynucleotide to a cell in accordancewith this embodiment of the present invention are known and common inthe art. Such techniques are described in Sambrook et al.(aforementioned), and this document explains a variety of standardexperimental manuals describing the aforementioned techniques in detail.In respect of this embodiment of the present invention, the vector is,for example, a plasmid vector, a single- or double-stranded phagevector, or a single- or double-stranded RNA or DNA viral vector. Such avector is introduced into a cell as a polynucleotide, and preferably asDNA by a common technique for the introduction of DNA or RNA into acell. When the vector is a phage or virus, the vector is preferablyintroduced to the cell as a packaged or sealed virus by a knowntechnique for infection and transduction. A viral vector may be of areplication-competent or defective type.

A preferable vector is a vector which expresses the polynucleotide orpolypeptide of the present invention in points. In general, such avector comprises a cis-action control region that is effective for theexpression in a host operably bound to the polynucleotide to beexpressed. When a suitable trans-action factor (for example, a group ofproteases involved with the post-translational processing such as signalpeptidase or Furin) is introduced in a host cell, it is supplied by ahost, a complementary vector, or the vector itself.

In a preferable embodiment, a vector provides specific expression. Suchspecific expression is an inducible one or realized only in a certaintype of cell. Alternatively, it is an inducible and cell-specificexpression. A particularly preferable inducible vector can induceexpression by an easily operable environmental factor such astemperature or a nutritional additive. Various vectors suitable for thisembodiment including a construction for the use in prokaryotic andeukaryotic cell hosts and an inducible expression vector are known, andpersons skilled in the art can commonly use them.

A genetically engineered host cell can be cultured in general nutrientmedium, and it is modified to be particularly suitable for activation ofpromoter, selection of transformant, or amplification of a gene. Ingeneral, it would be obvious to persons skilled in the art thatconventional culture conditions such as temperature or pH level for hostcells selected for the expression are suitable for the expression of thepolypeptide of the invention.

A wide variety of expression vectors can be used for expressing thepolypeptide of the present invention. Examples of these vectors includechromosome, episome, and virus-derived vectors. These vectors arederived from bacterial plasmid, bacteriophage, yeast episome, yeastchromosome element, or viruses such as baculovirus, papovavirus such assimian virus 40 (SV40), vaccinia virus, adenovirus, fowlpox virus,pseudorabies virus, or retrovirus. A vector derived from a combinationof the aforementioned, for example, a vector derived from plasmid andbacteriophage gene element, more specifically, a cosmid or phagemid, mayalso be used. They are used for the expression in accordance with thisembodiment of the present invention. In general, since polypeptides wereexpressed in hosts, any vector that is suitable for maintaining,multiplying, or expressing a polynucleotide can be used for theexpression according to the aforementioned embodiment. A suitable DNAsequence is inserted into a vector by various conventional techniques.In general, a DNA sequence for expression is bound to an expressionvector by cleavage of a DNA sequence and an expression vector having 1or more restriction endonucleases, and a restriction fragment is thenbound together using T4 DNA ligase. Restriction and ligation techniquesthat can be used for the above purpose are known and common to personsskilled in the art. With regard thereto, Sambrook et al.(aforementioned) very precisely describe another suitable method forconstructing an expression vector utilizing another technique known andcommon to persons skilled in the art.

A DNA sequence in the expression vector is operably bound to, forexample, a suitable expression-regulating sequence including a promoterto orient the mRNA transcription. A few examples of known representativepromoters are the phage lambda PI, promoter, E. coli lac, trp, trc, andtac promoters, SV40 early and late promoters, and the retrovirus LTRpromoter. Many promoters that are not described are suitable for the useaccording to the embodiment of the present invention, known, and moreeasily used as described in the examples of the present invention. Ingeneral, an expression construct comprises a ribosome binding site fortranslation in a transcription initiation or termination site or atranscribed domain. The coding region of the mature transcript that wasexpressed by the construct comprises the initiation AUG at theinitiation and termination codons located substantially at the terminusof polypeptide to be translated. In addition, the construct comprises aregulator region that regulates and induces the expression. In general,such a region is activated through the regulation of the repressorbinding site, transcription of an enhancer, or the like in accordancewith various conventional methods.

Vectors for multiplication and expression include selection markers.Such markers are suitable for multiplication, or they compriseadditional markers for the above-stated purpose. The expression vectorpreferably comprises one or more selection marker genes to providephenotypic traits for the purpose of selecting the transformed hostcell. A preferable marker includes dihydrofolate reductase- orneomycin-resistance with regard to eukaryotic cell culture. It hastetracycline- or ampicillin-resistance with regard to E. coli and otherbacterial cultures. A suitable vector comprising a DNA sequence and asuitable promoter or regulatory sequence as described herein areintroduced to a suitable host by various suitable known techniques forthe expression of the polypeptide of interest.

Representative examples of suitable hosts include: bacterial cells suchas E. coli, Streptomyces, and Salmonella typhimurium; fungal cells suchas a yeast cell; insect cells such as drosophila S2 and Spodoptera Sf9cells; and adhesive or floating animal or plant cells such as CHO, COS,Bowes melanoma cells, and SP2/0. Various hosts for expression constructsare known, and persons skilled in the art can easily select a host forexpressing polypeptides in accordance with this embodiment based on thedisclosure of the present invention.

More specifically, the present invention includes a recombinantconstruct, such as an expression construct comprising one or moresequences as mentioned above. The construct is a vector, such as aplasmid or viral vector comprising the sequence of the present inventioninserted therein. The sequence is inserted in a positive or negativedirection. In a preferable specific example thereof, the constructfurther has a regulatory sequence comprising a promoter or the like thatis operably bound to the sequence. Various suitable vectors andpromoters are known to persons skilled in the art, and there are manycommercially available vectors that are suitably used in the presentinvention.

Commercially available vectors are exemplified below. Vectors that arepreferably used for bacteria are pQE70, pQE60, and pQE-9 (Qiagen); pBSvector, PhageScript vector, Bluescript vector, pNH8A, pNH16a, pNH18A,and pNH46A (Stratagene); and ptrc99a, pKK223-3, pKK233-3, pDR540, andpRIT5 (Pharmacia). Examples of preferable eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1, and pSG (Stratagene) and pSVK3, pBPV, pMSG, andpSVL (Pharmacia). These vectors are commercially available for personsskilled in the art to be used in accordance with the embodiment of thepresent invention, and they are merely a list of known vectors. Forexample, other plasmids or vectors suitable for introducing,maintaining, multiplying, or expressing the polynucleotide orpolypeptide of the present invention can also be used in hosts inaccordance with this embodiment of the present invention.

A promoter region can be selected from a gene of interest using a vectorcomprising, for example, a candidate promoter fragment, i.e., a reportertranscription unit lacking a promoter region such as a chloramphenicolacetyl transferase (CAT) transcription unit located downstream ofrestriction sites for introducing promoter-containing fragments. Asknown to the public, the introduction of the promoter-containingfragment into the vector at the restriction site located upstream of thecat gene generates CAT activity that can be detected by standard CATassay. A vector that is suitable for this purpose is known and readilyavailable. Examples of such vectors are pKK232-8 and pCM7. Accordingly,the promoter for expressing the polynucleotide of the present inventionincludes not only a readily available known promoter but also a promoterthat can be readily obtained using a reporter gene in accordance withthe aforementioned technique.

Among them, according to the present invention, examples of knownbacterial promoters that are suitably used to express polynucleotidesand polypeptides are E. coli lad and lacZ promoters, T3 and T7promoters, gpt promoter, lambda PR and PL promoters, and trp and trcpromoters. Examples of suitable known eukaryotic promoters include theCytomegalovirus (CMV) immediate promoter, the HSV thymidine kinasepromoter, early and late SV40 promoters, a retrovirus LTR promoter suchas the Rous sarcoma virus (RoSV) promoter, and a metallothioneinpromoter such as the metallothionein-I promoter.

Selection of a vector and a promoter suitable for expression in a hostcell is a known technique. Techniques necessary for the construction ofexpression vectors, introduction of a vector in a host cell, andexpression in a host are common in the art. The present invention alsorelates to a host cell having the aforementioned construct. A host cellcan be a higher eukaryotic cell such as a mammalian animal cell, a lowereukaryotic cell such as a yeast cell, or a prokaryotic cell such as abacterial cell.

The construct can be introduced in a host cell by calcium phosphatetransfection, DEAE-dextran-mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,or other methods. These methods are described in a variety of standardlaboratory manuals, such as a book by Sambrook et al.

The construct in a host cell can be used by a conventional method, andit produces a gene product encoded by a recombinant sequence.Alternatively, a partial polypeptide of the present invention can besynthesized using a general peptide synthesizer. A mature protein can beexpressed under the control of a suitable promoter in a mammaliananimal, yeast, bacterial, or other cell. Also, such a protein can beproduced in a cell-free translation system with the use of RNA derivedfrom the DNA construct of the present invention. Suitable cloning andexpression vectors for prokaryotic and eukaryotic hosts are described inSambrook et al (aforementioned).

In general, a recombinant expression vector comprises: a replicationorigin; a promoter derived from a highly expressed gene to orient thetranscription of a downstream structural sequence; and a selectionmarker for bringing the cell into contact with a vector and isolatingthe vector-containing, cell. A suitable promoter can be induced from agene encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), α-factor, acid phosphatase, and heat shock protein. A selectionmarker includes E. coli ampicillin-resistant gene and S. cerevisiae trp1gene.

Transcription of DNA encoding the polypeptide of the present inventionusing a higher eukaryotic cell may be enhanced by inserting an enhancersequence in a vector. The enhancer is generally a cis-acting element forDNA for enhancing the promoter transcription activity in thepredetermined host cell. Examples of an enhancer include the SV40enhancer, the Cytomegalovirus early promoter/enhancer, the polyomaenhancer behind the replication origin, the β-actin enhancer, and theadenovirus enhancer.

The polynucleotide of the present invention encoding a heterologousstructural sequence of the polypeptide of the present invention isgenerally inserted in a vector by standard techniques in such a mannerthat it is operably bound to the expression promoter. The transcriptioninitiation site of the polypeptide is suitably located at the 5′ site ofthe ribosome binding site. The ribosome binding site is 5′ relative toAUG that initiates the translation of a polypeptide to be expressed. Ingeneral, an initiation codon starts from AUG and another open readingframe located between the ribosome binding site and initiation AUG isnot present. The termination codon is generally present at the terminusof the polypeptide, and the adenylation signal and the terminator aresuitably located at the 3′ end of the transcription region.

Regarding the secretion of the translated protein in the ER lumen, inthe cytoplasm, or to the extracellular environment, a suitable secretionsignal is incorporated in the expressed polypeptide. The signal may beendogenous or heterologous to the polypeptide.

Further, a prosequence subsequent to the signal sequence may beendogenous or heterologous (e.g., a preprosequence of another metalloprotease).

The polypeptide is expressed in a modified form such as a fusionprotein, and it includes not only a secretion signal but also anadditional heterologous functional region. Accordingly, an additionalamino acid, especially a charged amino acid region, or the like, isadded to the polypeptide to improve stability and storage stability inthe host cell during purification or subsequent operation and storage.Alternatively, a given region may be added to the polypeptide toaccelerate the purification. This type of region may be removed beforethe final preparation of polypeptides. Induction of secretion orexcretion, stability improvement, or facilitation of purification withthe addition of a peptide portion to the polypeptide is a techniquecommon and known in the art.

Examples of prokaryotic hosts that are suitable for multiplying,maintaining, or expressing the polynucleotide or polypeptide of thepresent invention include E. coli, Bacillus subtilis, and Salmonellatyphimurium. Various types of Pseudomonas, Streptomyces, andStaphylococcus are suitable hosts in this respect. Furthermore, variousother types of hosts known to persons skilled in the art can be alsoused. Representative examples of expression vectors that are useful forbacterial applications include, but are not limited to, the replicationorigin of bacteria derived from commercially available plasmid includinga selectable marker and a gene element of a known cloning vector pBR322(ATCC 37017). Examples of such commercially available vectors includepKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (PromegaBiotec, Madison, Wis., USA). These pBR322 (main chain) sections arecombined with a suitable promoter and structural sequences to beexpressed.

Host cells are suitably transformed and multiplied to the optimal cellconcentration. Thereafter, the selected promoter is induced by asuitable means (e.g., temperature shifting or chemical inducer), andcells are further cultured. Typically, cells are collected bycentrifugation and fractured by a physical or chemical means. Theresulting crude extract is further purified. Microbial cells used forthe protein expression can be fractured by any convenient means selectedfrom a freezing-thawing cycle, ultrasonication, mechanical fracture, andthe use of a cytolytic agent. These methods are known to persons skilledin the art.

Various cell lines for mammalian animal cell culture can be also usedfor the expression. An example of a cell line for mammalian animalexpression includes a monkey kidney fibroblast COS-6 cell described inGluzrnan et al., Cell 23: 175 (1981). Examples of other cells that arecapable of expressing compatible vectors include C127, 3T3, CHO, HeLa,human kidney 293, and BHK cells. Further, a floating myeloma cell linesuch as SP2/0 can be also used.

A mammalian animal expression vector comprises a replication origin, asuitable promoter and enhancer, a necessary ribosome binding site, apolyadenylation site, splice donor and acceptor sites, a transcriptiontermination sequence, and a 5′ franking untranscribed sequence necessaryfor expression. DNA sequences derived from the SV40 splice site and theSV40 polyadenylation site arc used for the non-transformed ortranscribed gene element of interest. An example thereof is a CAGexpression vector (H. Niwa et al. Gene, 108, 193-199 (1991)).

Based on the gene sequence of the above protease, a probe, primer, orantisense is designed by a common technique. The antisense technique canbe used for controlling gene expression by the use of antisense DNA orRNA or the formation of a triple helix. This technique is described in,for example, Okano, J., Neurochem., 56: 560 (1991);OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRCPress, Boca Raton, Fla. (1988). The triple helix formation is examinedin, for example, Lee et al., Nucleic Acids Research 6: 3073 (1979);Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251:1360 (1991). The method is based on the polynucleotide bond withcomplementary DNA or RNA. This enables the gene diagnosis or genetherapy.

For example, cells obtained from a patient are subjected to ex vivogenetic engineering using a polynucleotide such as polypeptide-encodingDNA or RNA. The resulting cells are then supplied to patients who shouldbe treated with polypeptides. For example, cells can be subjected to exvivo genetic engineering using a retrovirus plasmid vector comprisingRNA encoding the polypeptide of the present invention. Such a techniqueis known in the art, and the use thereof in the present invention isobvious according to the description given herein. Similarly, cells aresubjected to in vitro genetic engineering in accordance with aconventional process in respect of in vivo polypeptide expression. Forexample, the polynucleotide of the present invention is geneticallyengineered for expression in the replication-deficient retrovirus vectoras mentioned above. Subsequently, the retrovirus expression construct isisolated, introduced to a packaging cell, and transduced using aretrovirus plasmid vector comprising RNA encoding the polypeptide of thepresent invention. Thus, the packaging cell produces infectious viralparticles having a control gene. These producer cells are subjected toin vitro genetic engineering and then administered to patients to allowpolypeptides to be expressed in vivo. This administration method andother methods for administering polypeptides according to the presentinvention would be clearly understood by persons skilled in the artbased on the teaching of the present invention.

Examples of the aforementioned retrovirus, from which the retrovirusplasmid vector is derived, include, but are not limited to, Moloneymurine leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harveysarcoma virus, avian leukosis virus, gibbon leukemia virus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus. This type of vector comprises one or more promoters toexpress polypeptides. Examples of suitable promoters that can be usedinclude, but are not limited to, retrovirus LTR, SV40 promoter, CMVpromoter described in Miller et al., Biotechniques 7: 980-990 (1989),and other promoters (e.g., cell promoters such as a eukaryotic cellpromoter including, but not limited to, histone, RNA polymerase III, andβ-actin promoter). Examples of other viral promoters that can be usedinclude, but are not limited to, adenovirus promoter, thymidine kinase(TK) promoter, and B19 Parvovirus promoter. Persons skilled in the artcan readily select a suitable promoter based on the teaching of thepresent invention.

A nucleic acid sequence that encodes the polypeptide of the presentinvention is under the control of a suitable promoter. Examples ofsuitable promoters that can be used include, but are not limited to,adenovirus promoter such as adenovirus major late promoter, heterologouspromoter such as CMV promoter, respiratory syncytial virus (RSV)promoter, inducible promoter such as MMT promoter or metallothioneinpromoter, heat shock promoter, albumin promoter, ApoAI promoter, humanglobin promoter, viral thymidine kinase promoter such as herpes simplexthymidine kinase promoter, retrovirus LTR including the aforementionedmodified retrovirus LTR, β-actin promoter, and human growth hormonepromoter. A promoter may be of a native type that controls the geneencoding polypeptides. A retrovirus plasmid vector is used to transducethe packaging cell line to form a producer cell line.

Examples of packaging cells to be transfected include, but are notlimited to, PE501, PA317, Y-2, Y-AM, PA12, T19-14X, VT-19-17-H2, YCRE,YCRIP, GP+E-86, GP+envAm12, and the DAN cell line described in Miller,Human Gene Therapy 1: pp. 5-14 (1990).

A vector is transduced in a packaging cell by a means known in the art.Examples of such means include, but are not limited to, electroporation,the use of a liposome, and CaPO₄ precipitation. Alternatively, aretrovirus plasmid vector is sealed in a liposome or bound to a lipid tobe administered to a host. A producer cell line produces infectiousretrovirus vector particles comprising nucleic acid sequences encodingpolypeptides. Such retrovirus vector particles are used to transduceeukaryotic cells in vitro or in vivo.

The transduced eukaryotic cells express nucleic acid sequences encodingpolypeptides. Examples of eukaryotic cells that may be transducedinclude, but are not limited to, germinal stem cells, embryonalcarcinoma cells, hematopoietic stem cells, hepatic cells, fibroblasts,sarcoblasts, keratinocytes, endothelial cells, and bronchial epithelialcells.

The protease of the present invention, an antibody against thisprotease, an antagonist of this protease, an inhibitor, an agonist, anactivity modifier, or the like can be diluted with physiological saline,buffer, or the like to prepare a formulation. Thus, a pharmaceuticalcomposition can be obtained. The pH value of the formulation ispreferably between acidulous and neutral: close to the pH level of bodyfluid. The lower limit thereof is preferably between 5.0 and 6.4, andthe upper limit is preferably between 6.4 and 7.4. Alternatively, theformulation can be provided in a state that allows storage for a longperiod of time, e.g., in a lyophilized state. In such a case, theformulation can be used by being dissolved in water, physiologicalsaline, butler, or the like at a desired concentration level at the timeof use.

The formulation of the present invention may comprise apharmacologically acceptable additive, such as a carrier, excipient, ordiluent that is commonly used for pharmaceuticals, a stabilizer, orpharmaceutically necessary ingredients. Examples of a stabilizer includemonosaccharides such as glucose, disaccharides such as saccharose andmaltose, sugar alcohols such as mannitol and sorbitol, neutral saltssuch as sodium chloride, amino acids such as glycine, nonionicsurfactants such as polyethylene glycol, polyoxyethylene andpolyoxypropylene copolymers (Pluronic), polyoxyethylene sorbitan fattyacid ester (Tween), and human albumin. Addition thereof in amounts ofabout 1 to 10 w/v % is preferable.

An effective amount of the pharmaceutical composition of the presentinvention can be administered by, for example, intravenous injection,intramascular injection, or hypodermic injection in one or severalseparate dosages. The dosage varies depending on symptom, age, bodyweight, or other factors, and it is preferably 0.001 mg to 100 mg perdose.

Also, sense or antisense DNA encoding the protease of the presentinvention can be similarly prepared in a formulation to obtain apharmaceutical composition.

Further, the present invention includes methods for inhibiting plateletplug formation involved with heart infarction or brain infarction,methods for inhibiting arteriosclerosis, methods for preventingrestenosis, reembolization, or infarction involved with PTCA, methodsfor preventing reembolization involved with PTCR, and methods forpreventing platelet plug formation caused by HUS or O-157 through theadministration of the peptide, protein, and DNA of the presentinvention. Furthermore, the present invention includes the use of thepeptide, protein, and DNA of the present invention in the production ofpharmaceuticals for inhibiting platelet plug formation involved withheart infarction or brain infarction, pharmaceuticals for inhibitingarteriosclerosis, pharmaceuticals for preventing restenosis,reembolization, or infarction involved with PICA, pharmaceuticals forpreventing reembolization involved with PTCR, and pharmaceuticals forpreventing platelet plug formation caused by HUS or O-157.

The peptide or protein of the present invention is used as a leadingsubstance for amino acid modification. This enables the preparation of amolecule having activity that is different from that of the protease ofthe present invention. An example thereof is a variant molecule that canbe obtained by preparing an antagonist, which is obtained by preparing avariant deactivated through amino acid substitution between an aminoacid residue located around the active center in the metalloproteasedomain and another amino acid, separating a molecule recognition sitefrom a catalytic site, or varying one or both of these sites.

The use of an evaluation system for the vWF-cleaving activity describedherein enables the production of an antagonist/agonist. For example, aneffective antagonist can be a small organic molecule, a peptide, or apolypeptide. An example thereof is an antibody that is bound to thepolypeptide of the present invention, thereby inhibiting or eliminatingits activity.

Similarly, the use of the aforementioned evaluation system forvWF-cleaving activity enables the screening for a compound that iscapable of cleaving vWF. In such a case, the cleaving activity of thetest compound may be evaluated using the aforementioned evaluationsystem.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the vWF multimer structure and the pointcleaved by the vWF-cleaving protease.

FIG. 2 is a photograph showing the result of vWF multimer analysis(agarose electrophoresis).

FIG. 3 is a photograph showing the result of SDS-PAGE (5% gel) foranalyzing the caving activity of each plasma fraction under reducingconditions.

FIG. 4 is a photograph showing the result of SDS-PAGE (5% gel) foranalyzing the solubilized sample of fraction 1 (F1) paste undernon-reducing conditions.

FIG. 5 is a photograph showing the result of analyzing vWF-cleavingprotease fractions after being subjected to gel filtrationchromatography three times using the solubilized sample of F1 paste as astarting material. FIG. 5A is a chart showing gel filtrationchromatography, FIG. 5B shows the result of SDS-PAGE on fractions undernon-reducing conditions, and FIG. 5C shows the results of SDS-PAGE onvWF-cleaving activity under reducing conditions.

FIG. 6 is a photograph showing the results of analyzing vWF-cleavingprotease fractions in which the fraction collected by gel filtrationchromatography is purified by DEAE anion exchange chromatography. FIG.6A is a chart showing gel filtration chromatography, FIG. 6B shows theresult of SDS-PAGE (8% gel) on elution fractions under non-reducingconditions, and FIG. 6C shows the results of SDS-PAGE on vWF-cleavingactivity under reducing conditions. In FIG. 6C, three bands indicate anintact vWF molecule (remaining uncleaved), a vWF cleavage fragment, anda vWF cleavage fragment, respectively, as in FIG. 5C.

FIG. 7 is a photograph showing an electrophoresed fragment obtained whenthe vWF-cleaving protease fraction purified and concentrated by DEAFanion exchange chromatography is further purified by Biophoresis-basedSDS-PAGE (non-reducing conditions).

FIG. 8 is a photograph showing the result of electrophoresis on afraction obtained by further purifying a vWF-cleaving protease fractionby Biophoresis-based SDS-PAGE for analyzing vWF-cleaving proteaseactivity and SDS-PAGE on active fractions under reducing conditions.FIG. 8A shows the results of SDS-PAGE for analyzing vWF-cleavingprotease activity under non-reducing conditions, and FIG. 8B shows theresults of SDS-PAGE for analyzing active fractions under reducingconditions.

FIG. 9 relates to the identification of the vWF-cleaving protease gene,which is a diagram showing primers used for amplifying the gene fragmentfor a Northern blot probe.

FIG. 10 relates to the identification of the vWF-cleaving protease gene,which is a photograph showing Northern blot autoradiography. FIG. 10Ashows the results obtained when the protease-encoding gene is used as aprobe, and FIG. 10B shows the results obtained when a β-actin probe (RNAcontrol) is used.

FIG. 11 relates to the identification of the vWF-cleaving protease gene,and is a diagram showing the locations and the sequences of the primersused in the RACE experiments.

FIG. 12 is a diagram showing the locations of primers designed forcloning full-length cDNA.

FIG. 13 is a diagram showing a process for constructing a vectorcontaining full-length cDNA.

FIG. 14 is a photograph showing the expression in various cell lines(Western blotting under reducing conditions using anti-FLAG antibody,where the mock is prepared by inversely inserting a gene in anexpression vector). In FIG. 14, each lane shows the results using theindicated sample.

Lane 1: Mock (host: 293 cell)Lane 2: vWF-cleaving protease, cDNA+FLAG (host: 293 cell)Lane 3: Mock (host: HepG2 cell)Lane 4: vWF-cleaving protease, cDNA+FLAG (host: HepG2 cell)Lane 5: Mock (host: Hela cell)Lane 6: vWF-cleaving protease, cDNA+FLAG (host: Hela cell)

FIG. 15 is a photograph showing the activity assay of recombinantexpression protease (analysis of vWF-cleavage by SDS-PAGE undernon-reducing conditions, where the mock is prepared by inverselyinserting a gene in an expression vector). In FIG. 15, each lane showsthe results using the indicated sample.

Lane 1: Mock (host: Hela cell)Lane 2: Supernatant in which vWF-cleaving protease was expressed (host:Hela a cell)Lane 3: Mock (host: HepG2 cell)Lane 4: Supernatant in which vWF-cleaving protease was expressed (host:HepG2 cell)Lane 5: Mock (host: 293 cell)Lane 6: Supernatant in which vWF-cleaving protease was expressed (host:293 cell)Lane 7: Mock (host: BHK cell)Lane 8: Supernatant in which vWF-cleaving protease was expressed (host:BHK cell)Lane 9: Mock (host: COS cell)Lane 10: Supernatant in which v-cleaving protease was expressed (host:COS cell)Lane 11: Mock (host: CHO cell)Lane 12: Supernatant in which vWF-cleaving protease was expressed (host:CHO cell)

FIG. 16 is a photograph showing the result of Western blotting using anantibody established against the protease of the present invention,wherein Western blotting is carried out for various antiserums using the293 cell as a host and a recombinant vWF-cleaving protease. In FIG. 16,each lane shows the results obtained with the use of the indicatedsample.

Lane 1: Mouse antiserum (prepared by administering purified protein)Lane 2: Rabbit antiserum (prepared by hypodermically administering anexpression vector to a rabbit)Lane 3: Untreated rabbit antiserumLane 4: Rabbit antiserum (prepared by administering KLH-conjugatedpartial synthetic peptide)

FIG. 17 is a photograph showing the result of Western blotting using anantibody established against the protease of the present invention,wherein various samples derived from human plasma and recombinantexpression units are detected using rabbit antiserum obtained byadministering full-length cDNA of vWF-cleaving protease. In FIG. 17,each lane shows the results obtained with the use of the indicatedsample.

Lane 1: Partially purified sample derived from human plasmacryoprecipitateLane 2: Purified vWF-cleaving protease derived from human plasmaLane 3: Gel-filtrated FI paste sample obtained from pooled human plasmaLane 4: Recombinant vWF-cleaving protease (host: 293 cell)Lane 5: Recombinant vWF-cleaving protease (host: Hela cell)

FIG. 18 is a photograph showing the result of Western blotting using anantibody established against the protease of the present invention,wherein rabbit antiserum obtained by immunizing a rabbit with apartially synthesized peptide of the vWF-cleaving protease is used toconfirm the vWF-cleaving protease in healthy human plasma and that inthe plasma and gene recombinant vWF-cleaving protease of a TTP patient.In FIG. 18, each lane shows the results obtained with the use of theindicated sample.

Lane 1: Gel-filtrated FI paste sample obtained from pooled human plasmaLane 2: Normal human plasma 1Lane 3: Normal human plasma 2Lane 4: Normal human plasma 3Lane 5: TTP patients plasma 1Lane 6: TTP patients plasma 2Lane 7: Recombinant vWF-cleaving protease (host: 293 cell)Lane 8: Recombinant vWF-cleaving protease (host: Hela cell)

FIG. 19 is a diagram showing the result of ELISA using an antibodyprepared against the vWF-cleaving protease.

FIG. 20 is a photograph showing the result of SDS-PAGE (silver staining)analyzing each fraction of affinity purified vWF-cleaving protease usingan antibody under reducing conditions. In FIG. 20, each lane shows theresults obtained with the use of the indicated sample.

Lane 1: Applied culture supernatant (diluted 10-fold)Lane 2: Passed-through fractionLane 3: Washed fractionLane 4: Elution fraction

FIG. 21 is a photograph showing the results of evaluating neutralizingactivity using an antibody (SDS-PAGE for analyzing vWF-cleaving activityunder non-reducing conditions). In FIG. 21, each lane shows the resultsobtained with the use of the indicated sample.

Lane 1: vWF-cleaving protease solution: normal rabbit serum=1:1Lane 2: vWF-cleaving protease solution normal rabbit serum (diluted5-fold)=1:1Lane 3: vWF-cleaving protease solution: peptide-immunized rabbitserum=1:1Lane 4: vWF-cleaving protease solution: peptide-immunized rabbit serum(diluted 5-fold)=1:1Lane 5: vWF-cleaving protease solution: recombinant protein-immunizedrabbit serum=1:1Lane 6: vWF-cleaving protease solution: recombinant protein-immunizedrabbit serum (diluted 5-fold)=1:1Lane 7: vWF-cleaving protease solution: 10 mM EDTA=1:1Lane 8: vWF-cleaving protease solution: buffer only=1:1Lane 9: buffer (without vWF-cleaving protease): buffer=1:1

FIG. 22 is a diagram showing the construction of an expression vectorfor a molecular species lacking a C-terminal domain.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is hereafter described in detail with reference tothe following examples, although it is not limited to these examples.

Example 1 Preparation of vWF

A plasma cryoprecipitation (2g) was dissolved in 20 ml of buffer (0.01%Tween-80/50 mM Tris-HCl/100 mM NaCl, pH 7.4), and the resultant wassubjected to gel filtration using a Sephacryl S-500 HR Column (2.6×90cm, Amersham Pharmacia) to prepare vWF. Fractions were recovered at aflow rate of 2 ml/min in amounts of 6 ml each, vWF was analyzed byWestern blotting using a peroxidase-labeled rabbit anti-human vWFantibody (DAKO), and high-molecular-weight vWF fractions were pooled.The pooled fractions were subjected to multimer analysis using agaroseelectrophoresis as described below.

As shown in FIG. 1, vWF originally has a multimer structure in which vWFmonomer molecules are polymerized with each other at their N-terminusesor at their C-terminuses, and vWF is subjected to partial hydrolysis bythe vWF-specific cleaving protease. As a result of the analysis, asshown in FIG. 2, the purified vWF exhibited a multimer pattern based onagarose electrophoresis approximately equivalent to that in the plasmaof a healthy person (the ladder in the drawing shows the electrophoresispattern of vWF having a multimer structure, and the upper portionindicates vWF with advanced polymerization). This can prepare vWFcomprising substantially no impurities that degrade it, and thisfraction was used as a substrate when assaying the vWF-cleaving activityas described below.

Example 2 vWF-Cleaving Reaction

vWF-cleaving activity was assayed as follows. A sample comprising 10 mMbarium chloride (final concentration) was pre-incubated at 37° C. for 5minutes to activate protease. A buffer (15 to 20 ml, 1.5 M urea/5 mMTris-HCl, pH 8.0) was placed in a 50 ml Falcon Tube. Subsequently, amembrane filter (0.025 μm. Millipore) was floated therein, and 100 μl ofactivated sample prepared by mixing with 50 μl of vWF substrate solutionwas added. The resultant was allowed to stand in an incubator (37° C.)overnight and recovered from the filter on the next day. The recoveredsample was evaluated based on the vWF cleavage pattern as describedbelow in the “SDS-PAGE” section.

SDS-PAGE

SDS-5% polyacrylamide gel was autologously prepared and used. An SDSelectrophoresis buffer (2 in the presence or absence of a reducingagent, i.e., 2-mercaptoethanol) was added to 10 μl of the sampledescribed in the “vWF-cleaving activity assay” section, and theresultant was boiled for 3 minutes to prepare an electrophoresis sample.The gel was subjected to electrophoresis at 30 mA for 1 hour and thenstained with the Gel Code Blue Stain Reagent (PIERCE) utilizing CBBstaining. As shown in FIG. 1, activity is evaluated based on thedevelopment of a cleavage fragment and the presence or absence offragments remaining uncleaved under reducing or non-reducing conditions.This is more specifically described in Example 3 and FIG. 3 below.

Multimer Analysis Utilizing Agarose Electrophoresis Preparation of Gel,Electrophoresis

Low gelling temperature agarose (Type VII, Sigma) was added to 375 mMTris-HCl (pH 6.8) until a concentration of 1.4% was reached, followed byheating in a microwave oven to completely dissolve the gel. Thereafter,0.1% SDS was added, and the resultant was maintained at 56° C. Theresultant was made to flow into a gel mold and solidified by cooling at4° C. overnight (running gel). The next day, high gelling temperateagarose (SeaKem) was mixed with 375 mM Tris-HCl (pH 6.8) until aconcentration of 0.8% was reached, and dissolved by boiling in amicrowave oven. Thereafter, the resultant was maintained at 56° C.(stacking gel). The gel prepared on the previous day was cleaved,leaving a 10-cm fraction from the end uncleaved. The aforementioned gelwas made to flow into the cleaved portion, and the gel was made to keepflowing at 4° C. for at least 3 hours, followed by solidification.Pyronin Y was added to the sample described in the “vWF cleavingactivity assay” section above, and the gel was prepared undernon-reducing conditions without boiling. The gel was subjected toelectrophoresis at 10 mA for at least 24 hours using an SDS-PAGE buffer.

Western Blotting

After the electrophoresis, the gel was immersed in a transcriptionbuffer (0.005% SDS, 50 mM phosphate buffer, pH 7.4) for 10 minutes, andthe resultant was transferred to a nitrocellulose membrane using atranscription apparatus at 4° C. at 0.5 A overnight. Blocking wasperformed using a blotting solution (5% skim milk, PBS) for 30 minutes,and the gel was then allowed to react for at least 6 hours with theperoxidase-labeled rabbit anti-human vWF antibody (DAKO), which wasdiluted 1,000-fold with the blotting solution. Thereafter, the gel waswashed three times with the blotting solution and once with PBS, andcolor was developed using Konica Immunostain HRP-1000 (Konica), whichwas a substrate reaction solution for peroxidase. The purified vWFanalyzed in this assay was found to have been undegraded, but wassufficiently usable as a substrate in the present invention (FIG. 2).

Example 3 Preparation of vWF-Cleaving Protease

Plasma was subjected to ethanol fractionation developed by Cohn. Aprotease having high vWF-cleaving activity (one with high specificactivity) when protein levels in four fractions (i.e., starting plasma,cryoprecipitate, fraction I (FI) supernatant, and a paste) are madeequivalent to each other was selected. As shown in FIG. 3, the proteaseactivity was highest in the FI paste. The N-terminal sequence of thiscleavage fragment was analyzed, and as a result, activity derived fromthe cryoprecipitate and the FI paste were found to cleave the peptidebond between residues Tyr 842 and Met 843. Thus, the FI paste wasdetermined to be a main starting material for purification thereafter.

Solubilization of FI Paste

The FI paste was fractionated in fractions of 12g each and thencryopreserved. The paste was allowed to melt at 4° C. the day before itsuse. The next day, 120 ml of solubilizing buffer (0.05% azide, 50 mMTris-HCl (pH 7.4), 100 mM NaCl) was added at 10 mg/ml, and the mixturewas stirred at 37° C. for 2 hours. The product was centrifuged at 10,000rpm for 10 minutes, and the supernatant was then recovered, followed byfiltration with a prefilter, a 5.0 μm filter, and a 0.8 um filter inthat order. The resultant was determined to be a solubilized sample.FIG. 4 shows the result of SDS-PAGE of the solubilized sample.

Gel Filtration Chromatography of vWF-Cleaving Protease

The solubilized FI paste was applied to a Sephacryl S-300 HR Column(5×90 cm, Amersham Pharmacia) to conduct the first gel filtration. Abuffer comprising 0.05% azide, 50 mM Tris-HCl (pH 7.4), and 100 mM NaCl(hereinafter referred to as an “elution buffer”), which was the same asthe solubilizing buffer, was used. The flow rate was 5 ml/min,fractionation was initiated at 600 ml after the sample application, andfractions were recovered in amounts of 10 ml each. Fractions weresubjected to the vWF-cleaving reaction, and their activities were thenanalyzed by SDS-PAGE. Fractions that exhibited protease activity werepooled, and a small amount of saturated ammonium sulfate was graduallyadded dropwide thereto until a final concentration of 33% saturation wasreached. The mixture was further allowed to stand at 4° C. overnight.The next day, the product was centrifuged at 10,000 rpm for 10 minutes,and an active fraction of interest was recovered as a precipitate. Theprocedures comprising solubilization, gel filtration, and ammoniumsulfate precipitation were performed for 5 batches and the resultant wascryopreserved at −20° C.

The ammonium sulfate precipitates (2 to 3 batches) obtained by the firstgel filtration were dissolved in 50 ml of elution buffer, and passedthrough the Sephacryl S-300 HR Column (5×90 cm) in the same manner as inthe first gel filtration to perform the second gel filtration. Theelution buffer, conditions, operations, and the like were the same asthose in the first gel filtration. Fractions were subjected to thevWF-cleaving reaction, and their activities were then analyzed bySDS-PAGE. Fractions with activity were pooled, and ammonium sulfateprecipitation was similarly performed. These procedures were repeatedtwo times.

The ammonium sulfate precipitates (2 batches) obtained by the second gelfiltration were dissolved in 50 ml of elution buffer, and applied to theSephacryl S-300 HR Column (5×90 cm) in the same manner as in the firstand the second gel filtration to perform the third gel filtration. Theelution buffer, conditions, operations, and the like were the same asthose in the first and the second gel filtration. Fractions weresubjected to the vWF-cleaving reaction, and their activities were thenanalyzed by SDS-PAGE, followed by pooling. FIG. 5 shows SDS-PAGE foranalyzing these fractions and that for analyzing vWF-cleaving activity.Based on the patterns of gel filtration and the data showing activity,the protease of the present invention was found to be eluted in theregion between fraction 37 and fraction 47. Based on a separatelyconducted elution experiment for high-molecular-weight gel filtrationmarker (Amersham Pharmacia), this site of elution was deduced to have amolecular weight equivalent to 150 to 300 kDa. In this phase,considerable amounts of impurities were still present.

DEAE Anion Exchange Chromatography

The pooled fraction obtained by three gel filtration operations wassubjected to dialysis overnight with a buffer comprising 50 mM Tris-HCland 50 mM NaCl (pH 7.1). After the dialysis, anion exchangechromatography was performed using a 5 ml HiTrap DEAE-Sepharose FastFlow Column (Pharmacia) to conduct further purification andconcentration. Equilibrating and washing were performed using a buffercomprising 50 mM Tris-HCl (pH 7.1), and elution was performed using 0.25M NaCl. The flow rate was 5 ml/min, and 5 fractions of 5 ml each wererecovered and pooled. FIG. 6 shows the results of SDS-PAGE for analyzingelution fractions and those for analyzing vWF-cleaving activity. Basedon SDS-PAGE for activity assay, the protease of the present inventionhaving vWF-cleaving activity was considerably effectively concentratedin the elution fraction.

Fractionation Utilizing SDS-PAGE

The sample (5 ml) purified and concentrated by DEAE anion exchangechromatography was further concentrated to 0.5 ml using Centricon(molecular weight cut off: 10,000 Da, Amicon). The protease of thepresent invention was isolated by Biophoresis III (Atto Corporation)utilizing SDS-PAGE. In accordance with the Laemmli method (Nature, vol.227, 680-685, 1970), a buffer for electrophoresis tanks was prepared,and developed with 8% polyacrylamide gel to recover the electrophoresisfraction. FIG. 7 shows the result of SDS-PAGE for analyzing therecovered fractions. The buffer used for recovery was comprised of 50 mMTris-HCl and 10% glycerol (pH 8.8). As is apparent from FIG. 7, thisprocess according to the present invention has a high ability to produceseparation. FIG. 8 shows the results of analyzing activity of a fractionfurther purified by electrophoresis and the results of SDS-PAGE foranalyzing active fractions. The protease of the present invention can berecovered as an active molecule even after SDS-PAGE. When the activityof this protease in the plasma is determined to be 1 in terms ofspecific activity, a degree of purification of 30,000- to 100,000-foldwas deduced to be achieved based on the average protein content in theplasma (60 mg/ml).

Example 4 Partial Amino Acid Sequencing

The partial amino acid sequence of the isolated protease was determined.This protease, which was isolated using Biophoresis, was transferred toa PVDF membrane after SDS-PAGE by a conventional technique, air-dried,and then subjected to analysis using the automated protein sequencer(model 492; PE Applied Biosystems). As a result, the vWF-cleavingprotease of the present invention isolated under the above conditionswas found to comprise a polypeptide chain having a molecular weight of105 to 160 kDa in SDS-PAGE under reducing conditions. This protease wasalso found to have, as a partial sequence, Leu-Leu-Val-Ala-Val (SEQ IDNO: 1), and preferablyAla-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val (SEQ ID NO: 2).

Deduction of Isolated Protease Utilizing Bioinformatics

At present, bioinformatics enables the deduction of full nucleotidesequences encoding a polypeptide without substantial gene cloningthrough collation with information in the database accumulated in thepast (BIOINFORMATICS: A Practical Guide to the Analysis of Genes andProteins, edited by Andreas D. Baxevanis and B. F. Francis Ouellette).Based on the partial amino acid sequencing by the aforementioned process(Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val (SEQ ID NO:2)), the database was searched by the tblastn program. As a result, achromosome clone (AL158826) that was deduced to encode the protease ofthe present invention was identified by genomic database search.Further, a part of the protease of interest as the expressed sequencetag (EST) and a clone that was deduced to be a part of the polypeptideencoded by the aforementioned genome (AI346761 and AJ011374) wereidentified. The amino acid sequence as shown in SEQ ID NO: 3 or 7 wasdeduced based thereon to be an active vWF-cleaving protease site.

Example 5 Gene Identification

Synthesis of all the following synthetic primers was performed byGreiner Japan Co. Ltd. by request. Further, reagents used for generecombination were those manufactured by TAKARA. TOYOBO, and New EnglandBiolabs unless otherwise specified.

Preparation of a Gene Fragment as a Northern Blotting Probe

A sense primer (SEQ ID NO: 9) and an antisense primer (SEQ ID NO: 10)were prepared. PCR was carried out using Universal QUICK-Clone™ cDNA(Clontech), which was a mixture of cDNA derived from normal humantissue, as a template and TaKaRa LA Taq with GC rich buffer. A genesandwiched between these primers was amplified, and the amplifiedfragment was cloned using a TOPO TA Cloning™ kit (Invitrogen). DNAshaving the nucleotide sequence as shown in SEQ ID NO: 6 were isolatedfrom several clones.

A vector portion was removed from this cloned DNA by EcoRI digestion,separated and purified by agarose electrophoresis, and the resultant wasdetermined to be a template for preparing probes for Northern blotting.

Northern Blotting

The gene fragment prepared above was employed as a template to prepare aradioactive probe using [α-³²P]dCTP (Amersham Pharmacia) and a BcaBEST™labeling kit (TAKARA). Hybridization was carried out using the Human12-lane Multiple Tissue Northern Blots™ (Clontech) filter in accordancewith the method described in Molecular Cloning 2^(nd) Edition, pp.9.52-9.55. Detection was carried out by autoradiography. As shown inFIG. 10, mRNA encoding the protease was expressed mainly in the liver.The size of this mRNA was found to be more than 4.4 kb.

Isolation and Identification of Gene Encoding the Protease

As a result of Northern blotting, mRNA was found to be expressed mainlyin the liver. Thus, the protease gene of the present invention wasisolated and identified in accordance with the RACE technique usingnormal human liver-derived poly A⁺ RNA and MARATHON-READY™ cDNA(Clontech), which is a premade full length cDNA library ofadaptor-ligated ds cDNA ready for use.

More specifically, the first PCR was carried out as 5′ RACE using normalhuman liver-derived MARATHON-READY™ cDNA, which is a premade full lengthcDNA library of adaptor-ligated ds cDNA ready for use, in accordancewith the product's manual and using the AP-1 primer attached to the kitand antisense primers (SEQ ID NOs: 11 to 13) arbitrarily selected fromthe group of Gene Specific Primers (GSP) excluding the primer 1 locatedin the uppermost stream as shown in FIG. 11. Nested PCR (the second PCR)was then carried out using the AP-2 primer located in the inside thereofand the antisense primer located in the inside of the primer used forthe first PCR as shown in FIG. 11. Thereafter, TA cloning was carriedout. Genes were prepared from the developed colonies in accordance witha conventional technique (Molecular Cloning 2^(nd) Edition, pp.1.25-1.28), and nucleic acid sequences were decoded using an automaticDNA sequencer. The primer used for sequencing was the primer used forPCR or a primer located in the inside thereof. Further, the primer wasdesigned based on the sequence determined after serial decoding.

3′ RACE was started from normal human liver-derived poly A⁺ RNA usingthe 3′-Full RACE Core Set (TAKARA), and reverse transcription wascarried out in accordance with the attached manual using the attachedoligo dT primer. The band amplified by PCR using the sense primer (SEQID NO:14) located at “primer 2” in FIG. 11 and the attached oligo dTprimer was separated by agarose electrophoresis and extracted, followedby TA cloning. Genes were prepared from the developed colonies, andnucleic acid sequences were decoded using an automatic DNA sequencer. Aprimer used for sequencing was designed based on the sequence determinedafter serial decoding.

Example 6 Preparation of a Vector Comprising Full-Length cDNA 1

cDNA encoding the protein was subjected to one-stage PCR by, forexample, using a sense primer 1 (SEQ ID NO: 22) comprising an XhoIrestriction site and an initiation codon and an antisense primer 2 (SEQID NO: 23) comprising an SalI restriction site and a termination codon(see FIG. 12), using the aforementioned normal human liver-derivedMARATHON-READY™ cDNA, which is a premade full length cDNA library ofadaptor-ligated ds cDNA ready for use, as a template and the TaKaRa LATaq with GC rich buffer, followed by the aforementioned TA cloning.Thereafter, the full length of the product was confirmed using anautomatic DNA sequencer.

Example 7 Preparation of a Vector Comprising Full-Length cDNA 2

Restriction sites AccI and AvrII that cleaved cDNA only at one point onthe inner sequence of the cDNA (SEQ ID NO: 15) encoding the protein werefound. With the use thereof, full-length cDNA was divided into threefragments as shown in FIG. 12. A fragment 1 sandwiched between the senseprimer 1 (SEQ ID NO: 22) and the antisense primer 3 (SEQ ID NO: 24), afragment 2 sandwiched between the sense primer 4 (SEQ ID NO: 25) and theantisense primer 5 (SEQ ID NO: 26), and a fragment 3 sandwiched betweenthe sense primer 6 (SEQ ID NO: 27) and the antisense primer 2 (SEQ IDNO: 23) were provided, respectively, in each of the above threefragments. Each fragment was subjected to PCR using the aforementionednormal human liver-derived Marathon-Ready™ cDNA as a template and TaKaRaLA Taq with GC rich buffer, followed by the aforementioned TA cloning.The full length of the product was confirmed using an automatic DNAsequencer. Further, the pCR 2.1 vector included in the aforementioned TAcloning kit was subjected to self ligation, the ligation product wascleaved with XhoI/HindIII, ligated to a linker comprisingXhoI/AccI/AvrII/HindIII (prepared by annealing the synthetic DNA asshown in SEQ ID NO: 28 or 29), and the three aforementioned fragmentswere sequentially ligated in a conventional manner to bind them. Thus,cDNA comprising the entire region was prepared (see FIG. 13).

Example 8 Preparation of an Expression Vector Comprising Full-LengthcDNA: an Animal Cell Host

DNA obtained in Example 6 or 7 was digested with restriction enzymesXhoI/SalI, ligated to, for example the SalI site in the pCAG vector(Nivea, H. et al., Gene, vol. 108, 193-199), and the direction of theinsertion and the full-length sequence were confirmed using an automaticDNA sequencer.

Example 9 Transfection of an Expression Vector Comprising Full-LengthcDNA into an Animal Cell

The animal cell expression vector prepared in Example 8 was transfectedin the following manner using the 293 cell (human embryonic kidney cellline), the Hela cell, and the HepG2 cell. At the outset, cells weredisseminated at 1 to 3×10⁵ cells per 35 mm dish 24 hours before thetransfection. The next day, 2 μl of polyamine transfection reagent,TransIT (TAKARA), per μg of the expression vector, were added to 100 μlof a serum-free medium such as Opti-MEM to prepare a complex with DNA inaccordance with the instructions included with the reagent. Thereafter,the complex was added dropwise to the various types of previouslyprepared cells, and the resultants were incubated for 2 to 8 hours,followed by medium exchange. The medium was further exchanged three dayslater with the selective medium to which G418 had been added.Thereafter, medium was exchanged every three days to produce a stablyexpressed strain. An example thereof is shown in FIG. 14 as atemporarily expressed strain comprising an FLAG epitope tag at itsC-terminus. Detection was carried out by Western blotting using theanti-FLAG-M2 antibody (Kodack) and staining with anti-mouse Ig-alkalinephosphatase-labeled antibody system. The recombinant strain expressedusing cDNA as shown in this example exhibited a molecular size of about250 kDa under reducing conditions. This molecular size was also found inthe plasma of a healthy human (FIG. 18, Example 14 below). Severaldifferent molecular species of this protease are found to be present inthe human plasma, which could be caused by the presence of thealternative splicing products (SEQ ID NOs: 6 to 21) observed at the timeof gene cloning, difference in post-translational modification such assugar chain addition, or degradation during purification (described inExample 14 and in FIG. 17 of the present invention and Gerritsen et al.,Blood, vol. 98, 1654-1661 (2001)).

Subsequently, the vWF-cleaving activity of the recombinant strain wasconfirmed by the method described in Example 2 (FIG. 15). As a result,the human plasma-derived protease and the gene recombinant product ofthe present invention were found to exhibit the same vWF-cleavingactivities.

Example 10 Preparation of an Expression Vector Comprising Partial cDNA:an E. coli Host

Partial cDNA encoding the metalloprotease domain of the protein wassubjected to PCR using a sense primer comprising an NcoI restrictionsite and an initiation codon (SEQ ID NO: 30) and an antisense primercomprising an HindIII restriction site and a termination codon (SEQ IDNO: 31), the aforementioned normal human liver-derived MARATHON-READY™cDNA, which is a premade full length cDNA library of adaptor-ligated dscDNA ready for use, or the cDNA obtained in Example 6 or 7 as atemplate, and the TaKaRa LA Taq with GC rich buffer. The PCR product wasthen digested with NcoI/HindIII, ligated to the NcoI/HindIII digest ofan E. coli expression vector such as pUT1 (Soejima et al. J. Biochem.Tokyo, vol. 130, 269-277 (2001)), and transformed to the E. colicompetent cell JM 109 by a conventional technique. Several clones werecollected from the formed colony group, and genes were preparedtherefrom. Thereafter, the resulting genes were confirmed to be thegenes encoding the polypeptide, wherein the nucleic acid sequence of theinsertion site of the plasmid vector was equivalent to SEQ ID NO: 32 orsubstantially represented by SEQ ID NO: 33, using an automatic DNAsequencer.

Example 11 Expression of Partial cDNA-Containing Expression Vector in E.coli

An E. coli host with the expression vector constructed in Example 10introduced therein was precultured in 200 ml of LB medium comprising 50μg/ml ampicillin at 30° C. overnight. The resultant was sowed in afermenter comprising 8 liters of LB medium, and culture was conducted at30° C. until the turbidity at 600 nm became 0.2 to 0.5. Thereafter,isopropyl-1-thio-β-D-galactopyranoside was added to a finalconcentration of 1 mM, and the mixture was further cultured overnight toinduce the metalloprotease domain of the protein to be expressed. Thecultured E. coli were collected using a centrifuge (4° C. for 30minutes).

Subsequently, the collected E. coli pellet was resuspended in distilledwater, and lysozyme (final concentration: 0.6 mg/ml) was added thereto.The mixture was stirred at room temperature for 30 minutes, allowed tostand at 4° C. overnight, and cells were then destroyed. After theultrasonication, centrifugation was carried out using a centrifuge (4°C. for 20 minutes), and the pellet was recovered. The recovered pelletwas resuspended in a buffer comprising 50 mM Tris, 10 mM EDTA, and 1%Triton X-100 (pH 8.0). These procedures of centrifugation,ultrasonication, and resuspension were repeated several times, and thepellet was then resuspended in distilled water. Similarly, procedures ofcentrifugation, ultrasonication, and resuspension were repeated severaltimes to recover an inclusion body. This inclusion body was used as anantigen when producing an antibody.

Example 12 Isolation of Homologous Gene of Other Animal Species

The nucleic acid sequence as shown in SEQ ID NO: 15 was used as a probe,and a homology search was conducted using the BLASTN program at theGenomeNet WWW server (http://www.genome.ad.jp/). As a result, chromosomeclones AC091762 and AC090008 that were mapped at mouse chromosome 10were obtained. Based on these sequences, a mouse homolog of the proteaseof the present invention as shown in SEQ ID NO: 34 was deduced. A newprimer was designed from this sequence, and Northern blot analysis wasconducted by the technique used in isolating and identifying the geneencoding the human vWF-cleaving protease. Thus, the occurrence of thespecific expression in the liver was observed as with the case ofhumans. Further, normal mouse liver-derived poly A+ RNA andMARATHON-READY™ cDNA (Clontech) which is a premade full length cDNAlibrary of adaptor-ligated ds cDNA ready for use, were used to isolateand identify the protease gene of the present invention by the RACEtechnique as in the case of humans. As a result, the mouse homologousgene sequences of the protease as shown in SEQ ID NOs: 35 and 36 weredetermined.

Based on the thus determined mouse homologous partial sequence, theExon/Intron structure on the 5′ side of the aforementioned mousechromosome 10 was determined. In accordance with a conventionaltechnique (e.g., Gene Targeting: A Practical Approach First Edition,edited by A. L. Joyner, Teratocarcinomas and embryonic stem cell apractical approach), a targeting vector for knock-out (knock-in) micecan be prepared based thereon. This enabled the production of mutatedmice. Further, this protein can be subjected to recombinant expressionby a conventional technique.

Example 13 Production of an Antibody and Construction of a DetectionSystem for the Present Protease Using the Antibody

In accordance with a conventional technique (e.g., Current Protocols inMolecular Biology: Chapter 11 immunology, Antibody Engineering: APRACTICAL APPROACH, edited by J. McCAFFERTY et al. or ANTIBODYENGINEERING second edition, edited by Carl A. K. BORREBAECK), anexpression vector was administered to a mouse or rat. This expressionvector comprises a substance prepared by optionally binding an antigenprotein partially purified from human plasma or a synthetic peptidehaving a partial amino acid sequence thereof (e.g., a C-terminal peptidesequencePhe-Ser-Pro-Ala-Pro-Gln-Pro-Arg-Arg-Leu-Leu-Pro-Gly-Pro-Gln-Glu-Asn-Ser-Val-Gln-Ser-Ser(SEQ ID NO: 37), which was one isoform of the protease of the presentinvention) to an optimal carrier substance such as KLH (Cys was addedto, for example, the N- or C-terminus to facilitate KLH addition), theaforementioned gene recombinant protein, or a gene encoding thisprotein. Thus, a monoclonal antibody-expressing hybridoma wasestablished, and a polyclonal antibody (antiserum) was produced.

Subsequently, the antibodies prepared by the various aforementionedtechniques were used to detect the protease of the present invention byWestern blotting in accordance with a conventional technique (e.g.,Current Protocols in Molecular Biology: Chapter 10 analysis of proteins,Chapter 11 immunology). More specifically, the culture supernatant ofthe recombinant unit-expressing 293 cell obtained in the procedure asdescribed in Example 9 was subjected to SDS-PAGE under non-reducingconditions, transferred to a PVDF membrane, and confirmed using mouse orrabbit antiserum to confirm the expression of the geneticallyrecombinant unit (FIG. 16). As a result, a band that was deduced to bederived from the protease of the present invention was found in amolecular size range of 160 to 250 kDa. Subsequently, the protease ofthe present invention was detected using starting plasma or the like anda recombinant unit under non-reducing conditions. As a result, a bandwas found in 105 to 160 kDa or 160 to 250 kDa (FIG. 17). Also, a bandderived from a similar recombinant unit was detected in a monoclonalantibody established by immunizing a recombinant protein (clone No.CPHSWH-10).

Further, the C-terminal peptide sequencePhe-Ser-Pro-Ala-Pro-Gln-Pro-Arg-Arg-Leu-Leu-Pro-Gly-Pro-Gln-Glu-Asn-Ser-Val-Gln-Ser-Ser(SEQ ID NO: 37), which was one isoform of the protease of the presentinvention, was bound to KLH. The resultant was used as an immunogen toobtain a peptide antibody. With the use thereof, the protease of thepresent invention was detected from the plasma of healthy persons,plasma of TTP patients, or a culture supernatant of the recombinant unitunder reducing conditions. As a result, a band of approximately 250 kDathat was deduced to be a signal derived from the protease of the presentinvention was found, although it was not clear based on plasma derivedfrom some TTP patients (FIG. 18).

Furthermore, enzyme immunoassay (ELISA) constructed by combining theobtained antibodies enabled the preparation of a calibration curve thatis concentration-dependent at the culture supernatant level of therecombinant protein (FIG. 19). An example of ELISA is as follows. Theobtained mouse anti-vWF-cleaving protease antibody was immobilized onthe Maxisorp plate (Nunc), and 1/1, 1/2, and 1/4 diluents of the culturesupernatant of the vWF-cleaving protease-temporarily expressing 293cells were allowed to react in amounts of 100 μl/well (Mock supernatantas “0”). The plate was subjected to reaction, for example, at 37° C. for1 hour, and then washed with 0.05% Tween 20/TBS. Thereafter, the100-fold diluted rabbit anti-vWF-cleaving protease antibody was allowedto react in amounts of 100 μl/well, for example, at 37° C. for 1 hour,and the plate was washed with 0.05% Tween 20/TBS. The 1,000-fold dilutedperoxidase-labeled anti-rabbit Ig antibody (BioRad) was then allowed toreact in amounts of 100 μl/well, for example, at 37° C. for 1 hour, andthe plate was washed with 0.05% Tween 20/TBS. Thereafter, color wasdeveloped for a given period of time using a coloring substrate TMBZ,the reaction was terminated using 1M sulfuric acid as a terminationliquid, and the absorbance at 450 nm was assayed. The applicationthereof enabled the quantification of the protease of the presentinvention in a variety of specimens.

Example 14 Purification of the Protease Using an Antibody

The obtained antibody was bound to a suitable immobilization carrier toprepare an affinity column, and the resulting column was used to purifythe protease of the present invention. The affinity column was preparedby immobilizing an antibody using Cellulofine for NHS activation (ChissoCorporation) in accordance with the included instructions. The thusprepared swollen carrier (about 1 ml) was used to apply the culturesupernatant in which the recombinant gene had been expressed in the 293cell of the protease as described in Example 9. Thereafter, the columnwas washed with 50 mM Tris-HCl and 0.1M NaCl (pH 7.5, hereafter referredto as “TBS”), and elution was carried out using a urea-containing 0.1Mglycine buffer (pH 3). The eluted fraction was neutralized with 1MTris-HCl (pH 8.5) and then dialyzed against TBS. FIG. 20 shows theresults of SDS-PAGE analysis of the resulting purified protease. Also,the resulting purified fraction was found to have vWF-cleaving activity.The cleavage point of the vWF fragmented by this recombinant proteasewas found to be the position between residues Tyr 842 and Met 843 basedon the analysis of the N-terminal amino acid sequence of the fragment.Also established were clones (e.g. Clone Nos. CPHSWH-7.2 and 10) thatcould be similarly subjected to purification with the use of themonoclonal antibody prepared by the method as described in Example 13.

Subsequently, the partial amino acid sequence of the purified proteasewas determined. In accordance with a conventional technique, theprotease was subjected to SDS-PAGE, transferred to a PVDF membrane,air-dried, and then subjected to analysis using an automated proteinsequencer (model 492; PE Applied Biosystems). As a result, the proteasewas found to comprise the first five amino acids of SEQ ID NO: 2 as apartial N-terminal sequence. This sequence was congruous with theN-terminal sequence of the mature unit of the protease of the presentinvention that was deduced from the genetic construction.

Example 15 Neutralization of the Protease Activity Using an Antibody

Activity of the aforementioned rabbit polyclonal antibody to neutralizethe vWF-cleaving protease was evaluated. Normal rabbit serum, rabbitantiserum comprising the C-terminal peptide sequence,Phe-Ser-Pro-Ala-Pro-Gln-Pro-Arg-Arg-Leu-Leu-Pro-Gly-Pro-Gln-Glu-Asn-Ser-Val-Gln-Ser-Ser(SEQ ID NO:37) bound to KLH as an immunogen, and antiserum, the immunityof which had been induced by the protein expressed by the expressionvector as shown in Example 7 or 8, were respectively allowed topre-react at 37° C. for 1 hour with 1 to 10 of gene recombinantvWF-cleaving protease (approximated by the Bradford technique) at avolume ratio of 1:1. Alternatively, a 5-fold diluted antiserum wasallowed to pre-react under the above conditions with the protease at avolume ratio of 1:1. Thereafter, vWF-cleaving activity was evaluated bythe method described above. As a result, it was found that antiserum,which had activity of inhibiting the protease of the present invention,were prepared by immunizing the protein (FIG. 21). (antagonist activity)(a metalloprotease inhibitor, i.e., EDTA, was determined to be acontrol). This indicates the possibility of constructing an acquired TTPpatient-like model having a positive autoantibody against vWF-cleavingprotease as well as the simple possibility of producing a neutralizingantibody.

Example 16 Construction of C-Terminus Deleted Modification Unit

Based on the strategy shown in FIG. 22, the full-length vWF-cleavingprotease gene cloning vector (pCR 2.1 vWFCP) obtained in Example 6 or 7was used to add a variant lacking domains located in a positionfollowing the C-terminus (T1135stop, W1016stop, W897stop, T581stop, andQ449stop: each numerical value indicates the number of amino acidresidues between Met encoded by the initiation codon AGT and thetermination codon, and indicates a site comprising the FLAG epitope (DNAsequence: gactacaaggacgatgacgataagtga (SEQ ID NO: 47) and amino acidsequence: Asp Tyr Lys Asp Asp Asp Asp Lys (SEQ ID NO: 48)). Primers usedherein are as follows. “S” indicates a sense primer, and “AS” indicatesan antisense primer. Genes Stu I-S (SEQ ID NO: 38), Acc I-S (SEQ ID NO:39), Avr II-S (SEQ ID NO: 40), Q449stop-AS (SEQ ID NO: 41), T581stop-AS(SEQ ID NO: 42), W897stop-AS (SEQ ID NO: 43), W1016stop-AS (SEQ ID NO:44), T1135stop-AS (SEQ ID NO: 45), and full-length-AS (SEQ ID NO: 46)were prepared and incorporated in the pCAG expression vector inaccordance with the method as used in Examples 8 and 9. This expressionvector was introduced in the Hela cell. The primer pair shown at thebottom of the restriction map in the upper portion of FIG. 22 was usedto obtain PCR fragments (A) to (F). Each PCR fragment was ligated to pCR2.1 vWFCP. Further, the resultant was digested with StuI/SalI, andfragments (A) and (B) were digested with StuI/SalI and then ligated.These fragments were further digested with AccI, and fragment (C) wasalso digested with AccI, followed by ligation. The ligation product wasdigested with AvrII/SalI, and fragments (D), (E), and (F) were alsodigested with AvrII/SalI, followed by ligation. As a result, a variantlacking a region between the C-terminus and the position W897 was foundto have activity, although it was the result of qualitative analysis.Such a way of approach enables the identification of various functionaldomains. The design of molecules comprising these domains and having noprotease activity is considered to realize the design of antagonists oragonists.

INDUSTRIAL APPLICABILITY

The findings of the present invention have led to the possibility ofreplacement therapy for patients having diseases resulting fromdeficiency of a protease, such as thrombotic thrombocytopenic purpura.This also realizes the establishment of methods for gene cloning andefficient purification from serum or plasma. In particular, theinformation provided by the present invention enables gene recombinationbased on the obtained nucleotide sequence and stable production andprovision of the protease according to the present invention, which havebeen heretofore difficult to achieve. Also, these can be applied toreplacement therapy for TTP patients, inhibition of platelet plugformation involved with heart infarction or brain infarction, inhibitionof arteriosclerosis, prevention of restenosis, reembolization, orinfarction involved with PICA, prevention of reembolization involvedwith PTCR, and prevention of platelet plug formation caused by HUS orO-157. Diagnosis and therapy utilizing the gene encoding the protease ofthe present invention or an antibody thereagainst can be realized.

All publications cited herein are incorporated herein in their entirety.A person skilled in the art would easily understand that variousmodifications and changes of the present invention are feasible withinthe technical idea and the scope of the invention as disclosed in theattached claims. The present invention is intended to include suchmodifications and changes.

1. An antibody against a protease comprising a polypeptide chain havingthe amino acid sequence Leu-Leu-Val-Ala-Val, wherein the protease iscapable of cleaving a bond between residues Tyr-842 and Met-843 of vonWillebrand factor.
 2. The antibody according to claim 1, wherein theantibody inhibits or neutralizes the activity of a von Willebrand factorcleaving protease.
 3. A pharmaceutical composition or diagnostic agentcomprising an antibody against an isolated protease comprising apolypeptide chain having the amino acid sequence Leu-Leu-Val-Ala-Val(SEQ ID NO: 1), wherein the protease is capable of cleaving a bondbetween residues Tyr-842 and Met-843 of von Willebrand factor.